In-solution Y-chromosome capture-enrichment on ancient DNA libraries

Abstract Background As most ancient biological samples have low levels of endogenous DNA, it is advantageous to enrich for specific genomic regions prior to sequencing. One approach—in-solution capture-enrichment—retrieves sequences of interest and reduces the fraction of microbial DNA. In this work, we implement a capture-enrichment approach targeting informative regions of the Y chromosome in six human archaeological remains excavated in the Caribbean and dated between 200 and 3000 years BP. We compare the recovery rate of Y-chromosome capture (YCC) alone, whole-genome capture followed by YCC (WGC + YCC) versus non-enriched (pre-capture) libraries. Results The six samples show different levels of initial endogenous content, with very low (< 0.05%, 4 samples) or low (0.1–1.54%, 2 samples) percentages of sequenced reads mapping to the human genome. We recover 12–9549 times more targeted unique Y-chromosome sequences after capture, where 0.0–6.2% (WGC + YCC) and 0.0–23.5% (YCC) of the sequence reads were on-target, compared to 0.0–0.00003% pre-capture. In samples with endogenous DNA content greater than 0.1%, we found that WGC followed by YCC (WGC + YCC) yields lower enrichment due to the loss of complexity in consecutive capture experiments, whereas in samples with lower endogenous content, the libraries’ initial low complexity leads to minor proportions of Y-chromosome reads. Finally, increasing recovery of informative sites enabled us to assign Y-chromosome haplogroups to some of the archeological remains and gain insights about their paternal lineages and origins. Conclusions We present to our knowledge the first in-solution capture-enrichment method targeting the human Y-chromosome in aDNA sequencing libraries. YCC and WGC + YCC enrichments lead to an increase in the amount of Y-DNA sequences, as compared to libraries not enriched for the Y-chromosome. Our probe design effectively recovers regions of the Y-chromosome bearing phylogenetically informative sites, allowing us to identify paternal lineages with less sequencing than needed for pre-capture libraries. Finally, we recommend considering the endogenous content in the experimental design and avoiding consecutive rounds of capture, as clonality increases considerably with each round

Experimental conditions improving in-solution target enrichment for ancient DNA

High-throughput sequencing has dramatically fostered ancient DNA research in recent years. Shotgun sequencing, however, does not necessarily appear as the best-suited approach due to the extensive contamination of samples with exogenous environmental microbial DNA. DNA capture-enrichment methods represent cost-effective alternatives that increase the sequencing focus on the endogenous fraction, whether it is from mitochondrial or nuclear genomes, or parts thereof. Here, we explored experimental parameters that could impact the efficacy of MYbaits in-solution capture assays of ~5000 nuclear loci or the whole genome. We found that varying quantities of the starting probes had only moderate effect on capture outcomes. Starting DNA, probe tiling, the hybridization temperature and the proportion of endogenous DNA all affected the assay, however. Additionally, probe features such as their GC content, number of CpG dinucleotides, sequence complexity and entropy and self-annealing properties need to be carefully addressed during the design stage of the capture assay. The experimental conditions and probe molecular features identified in this study will improve the recovery of genetic information extracted from degraded and ancient remains.No Full Tex

The genomic history of the Aegean palatial civilizations

The Cycladic, the Minoan, and the Helladic (Mycenaean) cultures define the Bronze Age (BA) of Greece. Urbanism, complex social structures, craft and agricultural specialization, and the earliest forms of writing characterize this iconic period. We sequenced six Early to Middle BA whole genomes, along with 11 mitochondrial genomes, sampled from the three BA cultures of the Aegean Sea. The Early BA (EBA) genomes are homogeneous and derive most of their ancestry from Neolithic Aegeans, contrary to earlier hypotheses that the Neolithic-EBA cultural transition was due to massive population turnover. EBA Aegeans were shaped by relatively small-scale migration from East of the Aegean, as evidenced by the Caucasus-related ancestry also detected in Anatolians. In contrast, Middle BA (MBA) individuals of northern Greece differ from EBA populations in showing ∼50% Pontic-Caspian Steppe-related ancestry, dated at ca. 2,600-2,000 BCE. Such gene flow events during the MBA contributed toward shaping present-day Greek genomes.We thank the INCD (https://incd.pt/) for use of their computing infrastructure, which is funded by FCT and FEDER ( 01/SAICT/2016 022153 ).C.P., E.G., A.S., L.W., and J. Burger acknowledge the support of the European Union and the General Secretariat of Research and Innovation-GSRI, Ministry of Development & Investments in Greece, and the Federal Ministry of Education and Research-BMBF in Germany under the Bilateral Cooperation Program Greece – Germany 2017 (project BIOMUSE-0195 ). O.L. and O. Dolgova acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, Centro de Excelencia Severo Ochoa, CERCA Programme/Generalitat de Catalunya, Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement, as well as co-financing with funds from the European Regional Development Fund by the Spanish Ministry of Science and Innovation corresponding to the Programa Operativo FEDER Plurirregional de España (POPE) 2014-2020, and by the Secretaria d’Universitats i Recerca, Departament d’Empresa i Coneixement of the Generalitat de Catalunya corresponding to the Programa Operatiu FEDER de Catalunya 2014-2020. F.C., C.E.G.A., S.N., D.I.C.D., L.A., B.S.d.M., Y.O.A.C., F.M., J.V.M.-M., and A.-S.M. were supported by the Swiss National Science Foundation (SFNS) and a European Research Council (ERC) grant to A.-S.M. M.U., S.T., D.U.-K., and C.P. were co-financed by the EU Social Fund and the Greek national funds research funding program ARISTEIA II ( project-3461 ). C.P., E.G., A.S., L.W., and J. Burger were co-financed by the Greek-German bilateral cooperation program 2017 (General Secreteriat for Research and Innovation, Ministry of Development and Investments, Greece, and Federal Ministry of Education and Research - BMBF, Germany) project BIOMUSE-0195 funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020 ) and co-financed by Greece and the European Union (EU Social Fund and European Regional Development Fund). E.K. was funded by the Greek State Scholarships Foundation (IKY). O. Delaneau is funded by a SNSF (project grant PP00P3_176977 ). V.C.S. was supported by Portuguese Foundation for Science and Technology (FCT-Fundação para a Ciência e Tecnologia) through funds granted to cE3c ( UIDB/00329/2020 ) and individual grant CEECIND/02391/2017 . O.L. was supported by a Ramón y Cajal grant from the Spanish Ministerio de Economia y Competitividad (MEIC) (RYC-2013-14797), a PGC2018-098574-B-I00 (MEIC/FEDER) grant, and the support of Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya ( GRC 2017 SGR 937 ). O. Dolgova was supported by a PGC2018-098574-B-I00 (MEIC/FEDER) grant. J.D.J. was funded by National Institutes of Health grants R01GM135899 and R35GM13938